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Fig. 4. Localisation and effect of overexpressed Btn1p. (A) Cells deleted for btn1 and expressing GFP-Btn1 for 18 hours (green, left top panel) were labelled with FM4-64 (red, left middle panel) to show the steady state location of GFP-Btn1. At time 0 the promoter nmt42 was repressed by the addition of thiamine and the location of GFP-Btn1 followed and compared with FM4-64 staining of vacuoles at hourly intervals. GFP-Btn1 was first visible in compartments adjacent to the poles or septum of the cells, then over a 3-hour period trafficked through these small pre-vacuolar compartments to the vacuolar membrane. Bar, 10 µm. (B) In cells deleted for btn1 and expressing GFP-Btn1 (green, top panel), the uptake of FM4-64 after 3 minutes and 30 minutes (red, middle panel) was compared with the location of GFP-Btn1. GFP-Btn1 colocalised with compartments that were stained with FM4-64, indicating trafficking through the endocytic route. (C) Correlation of vacuole size with GFP-Btn1 expression levels in btn1{Delta} cells. Vacuoles were visualised by FM4-64 staining and GFP expression by densitometry. Cells having the highest levels of expression of GFP-Btn1 had the smallest vacuoles, and vice versa. (D) Time course of vacuole size and GFP-Btn1 expression in btn1{Delta} cells. Vacuole size (open circles) was visualised by FM4-64 staining and GFP expression (filled circles) by fluorimetry. As overall levels of GFP-Btn1 increased following induction of expression, the average vacuolar size decreased.





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